mechanoLight: A genetically encoded fluorescent biosensor for visualizing 5-HT2AR mechanotransduction in live cells
7 affiliations
- 1Department of Converging Science and Technology, Kyung Hee University, Seoul 02447, Republic of Korea
- 2Department of Pharmacology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
- 3Neuroscience Research Institute, Medical Research Institute, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
- 4Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
- 5Brain Korea 21 FOUR Biomedical Science Project, Seoul National University College of Medicine, Seoul 03080, Republic of Korea
- 6Biomedical Research Division, Korea Institute of Science and Technology, Seoul 02792, Republic of Korea
- 7Seoul National University Bundang Hospital, Seongnam 13620, Republic of Korea
Abstract
G protein–coupled receptors (GPCRs) are pivotal sensors of the mechanical environment, yet how they sense and transduce physical stimuli into biochemical signals within live cells remains largely elusive. Here we introduce mechanoLight, a genetically encoded red fluorescent biosensor designed to report the mechanical activation of the 5-hydroxytryptamine 2A receptor (5-HT2AR). By incorporating a circularly permuted mApple (cp-mApple) into the third intracellular loop of 5-HT2AR with the optimized linker (m-linker), mechanoLight selectively detects conformational changes of 5-HT2AR in response to fluid shear stress while exhibiting minimal sensitivity to 5-HT. In sharp contrast, psychLight2, the previously developed green 5-HT2AR biosensor, primarily detects ligand-induced 5-HT2AR activation, suggesting that mechanoLight and psychLight2 capture distinct conformational modes of 5-HT2AR activation. Integrated analysis of linker-swapping experiments, structural modeling, and molecular dynamics simulations revealed that the flexibility of the m-linker underlies the mechanosensitivity of mechanoLight. We further extend this strategy to other mechanosensitive GPCRs, enabling visualization of their mechanoreactivity in live cells. The mechanoLight signal reflects the mechanosensitivity of 5-HT2AR, as shear stress-induced 5-HT2AR activation triggers Gαq-dependent signaling pathways, including phospholipase C (PLC) activation, intracellular Ca2+ mobilization, and ERK phosphorylation. Mutational analysis further identifies N-terminal glycosylation and helix 8 as structural determinants of 5-HT2AR mechanotransduction. Together, our findings establish 5-HT2AR as a mechanotransducer and introduce mechanoLight as a powerful tool for deciphering the molecular principles of GPCR mechanotransduction in live cells.
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BibTeX
@article{baek2026mechanolight,
title = {{mechanoLight: A genetically encoded fluorescent biosensor for visualizing 5-HT2AR mechanotransduction in live cells}},
author = {In-Yeop Baek and Hyunbin Kim and Duwan Kwon and Hahnbeom Park and Jihye Seong},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2026},
month = oct,
doi = {10.1073/pnas.2610816123},
url = {https://doi.org/10.1073/pnas.2610816123},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - mechanoLight: A genetically encoded fluorescent biosensor for visualizing 5-HT2AR mechanotransduction in live cells
AU - In-Yeop Baek
AU - Hyunbin Kim
AU - Duwan Kwon
AU - Hahnbeom Park
AU - Jihye Seong
PY - 2026
DA - 2026/10/19
JO - Proceedings of the National Academy of Sciences of the United States of America
DO - 10.1073/pnas.2610816123
UR - https://doi.org/10.1073/pnas.2610816123
AB - G protein–coupled receptors (GPCRs) are pivotal sensors of the mechanical environment, yet how they sense and transduce physical stimuli into biochemical signals within live cells remains largely elusive. Here we introduce mechanoLight, a genetically encoded red fluorescent biosensor designed to report the mechanical activation of the 5-hydroxytryptamine 2A receptor (5-HT2AR). By incorporating a circularly permuted mApple (cp-mApple) into the third intracellular loop of 5-HT2AR with the optimized linker (m-linker), mechanoLight selectively detects conformational changes of 5-HT2AR in response to fluid shear stress while exhibiting minimal sensitivity to 5-HT. In sharp contrast, psychLight2, the previously developed green 5-HT2AR biosensor, primarily detects ligand-induced 5-HT2AR activation, suggesting that mechanoLight and psychLight2 capture distinct conformational modes of 5-HT2AR activation. Integrated analysis of linker-swapping experiments, structural modeling, and molecular dynamics simulations revealed that the flexibility of the m-linker underlies the mechanosensitivity of mechanoLight. We further extend this strategy to other mechanosensitive GPCRs, enabling visualization of their mechanoreactivity in live cells. The mechanoLight signal reflects the mechanosensitivity of 5-HT2AR, as shear stress-induced 5-HT2AR activation triggers Gαq-dependent signaling pathways, including phospholipase C (PLC) activation, intracellular Ca2+ mobilization, and ERK phosphorylation. Mutational analysis further identifies N-terminal glycosylation and helix 8 as structural determinants of 5-HT2AR mechanotransduction. Together, our findings establish 5-HT2AR as a mechanotransducer and introduce mechanoLight as a powerful tool for deciphering the molecular principles of GPCR mechanotransduction in live cells.
SN - 0027-8424
SN - 1091-6490
ER -